Program Director/Principal Investigator (Last, First, Middle): Jose, PedfO A. PROJECT SUMMARY {See instructions): The Dl receptor (DIR) and DSR regulate ion transport in the renal proximal tubule, thick ascending limb, and other nephron segments. In the previous cycle, we showed that that SNX5 and SNX1 are important in the intemalization of DIR and DSR, respectively. Moreover, selective renal deletion of SNX1 or SNXS increases blood pressure in mice and rats. In human renal proximal tubule cells, SNXS is expressed in non- lipid rafts while DIR, GRK4, and PP2A-catalytic subunit/ PPP2R2C2, and VPS 26 are in lipid and non-lipid rafts. Dl-like receptor stimulation shifts DIR, GRK4, PP2A catalytic subunit/PPP2R2C2, and VPS 26 to non-lipid rafts where SNXS is located. We hypothesize that the assembly of DIR with the signaling complex in membrane microdomains is govemed by SNXS. SNXS may act as a chaperone in the intracellular trafficking of DIR after agonist stimulation. This hypothesis will be tested in human renal proximal tubule cells; mouse proximal and distal convoluted tubule celis will be studied also because the role of these proteins on renal function and blood pressure will be tested in genetically manipulated mice. Specific aim 1 will test the hypothesis that SNXS is important in the assembly of DIR, PP2A complex, and GRK4 in non- lipid raft membrane microdomains, as well as the intemalization of this complex after agonist stimulation and subsequent recycling. Specific aim 2 will test the hypothesis that Dl R is linked to adenylyl cyclase (AC) III and AC VI and DSR is linked to AC IV in specific membrane microdomains in renal proximal tubules. In non- renal proximal tubule cells, D1R is linked to AC V. However, AC V is not expressed in renal proximal tubules. Specific aim 3 will test the hypothesis that PPP2R2C, SNX1, and SNXS are important in the regulation of blood pressure. These in vitro and in vivo studies may give important information on how specificity is confened on G protein-coupled receptor action, related to second messengers, sodium transport, and subsequently hypertension. At least two hypotheses are novel: 1) that SNXS is important in the assembly and signaling of DIR, PP2A, AC III and AC VI and GRK4 while DSR is linked to AC IV. At least two methods are novel: selective renal gene silencing in mice and inducible/reversible knockout. RELEVANCE (See Instructions): The cause and mechanisms leading to essential hypertension are still not well understood.These studies have important implication in hypertension because GRK4 gene variants are associated with hypertension; of all the genes thought to be important in hypertension, only the human GRK4 gene variants have been shown to produce hypertension in mice. SNX1 and PP2R2C gene variants are associated with hypertension in Caucasian Americans PROJECT/PERFORMANCE SrrE(S) (if additional space Is needed, use